Gene therapy involves the introduction of genetic material into the cells of an organism to treat or prevent a disease. The material transferred can be from one to a few genes in size. Since many hereditary diseases are caused by defects in single genes, there are many potential applications of this technique to the treatment of disease in humans and other animals. In addition, gene therapy is useful in the treatment and prevention of acquired diseases, such as infectious diseases and cancer.
Gene therapy can be directed to either an animal's germ line or somatic cells. For ethical and practical considerations, only somatic cell therapy is being pursued for humans. A variety of cell types have been targeted in somatic cell gene therapy systems, including hematopoietic cells, skin fibroblasts and keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, and lymphocytes, each with varying success.
The hematopoietic stem cells are a primary target for gene therapy because of well developed procedures for bone marrow transplantation, the many types and wide distribution of hematopoietic cells, and the existence of many diseases that affect hematopoietic cells. However, hematopoietic stem cells have proven difficult to infect in sufficient numbers with gene vectors, and differentiated hematopoietic cells suffer from short term expression of the gene of interest. Miller, Blood 76: 271-278 (1990), and Miller, Nature 357: 455-460 (1992). Gene transfer methods into muscle cells also demonstrate relatively short term expression and/or expression at low levels, and do not allow for efficient transport of the gene product into the bloodstream. Vascular endothelial cells have the advantage of direct access to the circulation, but are limited by the number of endothelial cells present in large vessels, since the layer of endothelial cells that line the vessels is only one cell thick. Gene transfer into lymphocytes is limited by the finite lifespan of the cells and thus requires repetitive administration to treat an ongoing disease. Gene transfer experiments into skin fibroblasts and keratinocytes have also been hampered by lack of prolonged improvement or insufficient levels of expressions.
Methods for gene therapy involving the liver have relied on gene transfer ex vivo, i.e., into hepatocytes which have been removed from a patient and are then reimplanted into the liver, or gene transfer in vivo, i.e., gene transfer directly into the liver. For ex vivo methods, gene transfer into cells must occur at high efficiency to obtain suitable numbers of cells for transplantation, because primary cultures of hepatocytes cannot be expanded.
Using the ex vivo approach, long term gene expression from transduced hepatocytes has been accomplished with retroviral vectors. The efficiency of transduction is relatively low, however, and the protein may not be expressed in therapeutically or prophylactically effective amounts. In one ex vivo method approximately 20% of a patient's liver is surgically removed, the cells are then transduced with the retroviral vector, and then implanted back into the patient. The retrovirus has been shown to infect only dividing cells. Miller et al., Mol. Cell. Biol. 10:4239-4242 (1990). This approach suffers from obvious disadvantages of surgical procedures and a low efficiency of transduction and expression of the gene product of interest.
The direct in vivo approach involves performing a two-thirds partial hepatectomy followed by portal vein infusion of the vector. The removal of the majority of the liver is needed to stimulate liver regeneration so that the retrovirus will integrate into the cell's chromosome. As with the ex vivo approach, this method suffers from requiring a major surgical procedure and under the best of conditions only about 1% of the liver mass contains the genetically modified vectors.
As an alternative to retroviral-mediated hepatic gene therapy, the adenovirus presents a transfer vector that can infect nonreplicating cells at high efficiency. Unfortunately, adenoviral DNA remains extra-chromosomal and thus is slowly lost from transduced hepatocytes over a period of several months. Li et al., Human Gene Ther. 4: 403-409 (1993); Kay et al., Proc. Natl. Acad. Sci.USA 91: 2353-2357 (1994). Additionally, a substantial portion of the adenovirus is taken up by organs and tissues other than the liver, which may raise issues of safety. (Smith et al., 1993 and Kay et al., ibid.). And, as adenovirus stimulates the production of neutralizing antibodies in an infected host, patients who have been naturally infected with adenovirus may be resistant to gene therapy using this vector, or secondary transductions may be prevented by the presence of antibodies produced in response to a primary transduction. (Smith ibid., Kay, ibid.).
The liver is a desirable target for somatic gene transfer because it is a large organ that is responsible for the synthesis, processing and secretion of many circulating proteins, including many of the plasma coagulation proteins. Because of the liver's involvement in many diseases of medical importance much effort has focused on replacing diseased livers by transplantation or, due to a severe shortage of donor livers, by implantation of healthy liver cells. Typically, however, implanted hepatocytes have made only small and temporary contributions to liver function.
Transgenic animal technology has been employed to create and analyze models of diseases affecting many organs, including the liver. A variety of transgenes have been reported to be associated with liver lesions, including urokinase-type plasminogen activator (uPA). In mice containing a albumin-urokinase transgene the hepatocyte-targeted expression of the uPA gene created a functional liver deficit. The uPA gene caused the fatal hemorrhaging of newborn mice, and survivors displayed hypofibrinogenemia and unclottable blood. It was thus concluded that any injury sufficient to initiate bleeding was rapidly fatal in affected mice. Heckel et al., Cell 62: 447-456 (1990). Surviving mice did show a gradual decrease in the level of plasma uPA activity, accompanied by a restoration of clotting function within one to two months. This was explained by a report that the uPA was cytotoxic for hepatocytes and that inactivation of transgene expression by DNA rearrangement in isolated hepatocytes in Alb-uPA mice was followed by repopulation of the entire liver by cells that no longer produce uPA. Sandgren et al., Cell 66: 245-256 (1991). The uPA transgene-expressing hepatocytes were at a selective disadvantage relative to hepatocytes (native or non-native) that were not expressing the transgene. Thus, production of uPA by the liver kills hepatocytes over time, and the gene encoding uPA has been used to impair native liver function and stimulate the repopulation of liver with non-native cells. See Brinster et al., PCT Publication WO 94/02601, and Rhim et al., Science 263: 1149-1152 (1994).
There remains a significant need in the art for methods of somatic gene therapy that use the liver for efficient expression of gene product in therapeutically useful quantities and duration. Desirably, these methods should (a) avoid the necessity for surgically removing a large portion of the liver, (b) enhance the yield and recovery of transduced hepatocytes without compromising viability; and (c) be independent of the particular disease being treated. Quite surprisingly, the present invention fulfills these and other related needs.